1. Technical Field
This invention relates to the field of molecular biology and plant fungal disease. In particular, the invention relates to the discovery of a fungal protein that is essential for pathogenicity in Magnaporthe grisea, a plant pathogen.
2. Description of the Background Art
The ascomycete Magnaporthe grisea causes disease in several monocot plant species, including blast disease in the important crop rice, and represents a model system to study fungal-plant interaction. See Ou et al., Rice Diseases, Commonwealth Mycological Institute, Surrey, 1985; Valent et al., “Rice blast as a model system for plant pathology” Phytopathology 80:33-36, 1990. Rice blast disease is an important problem in rice cultivation and a major threat to food security around the world, causing very significant crop losses annually. M. grisea also can cause diseases in a number of other important cereal crops such as wheat, rye and barley, and also in weed and turf grasses.
The asexual life cycle of M. grisea involves sporulation of the fungal hyphae to produce fruiting structures called conidia. The conidia contain many spores, which germinate in a suitable environment on a host plant, and develop to form a specialized infection structure termed an appressorium. These appressoria penetrate the plant cell by producing a penetration peg that is forced through the plant cell wall to begin infection. Upon entry of the fungus into the host cells, the fungus proliferates by forming penetration and infectious hyphae that establish and spread the disease lesions. See Hamer and Talbot, “Infection-related development in the rice blast fungus Magnaporthe grisea.” Curr. Opin. Microbiol., 1:693-697, 1998; Talbot, “On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea” Annu. Rev. Microbiol. 57:177-202, 2003. Infection of immature plants often is fatal. While mature plants usually survive, crop yield is considerably curtailed due to reduced photosynthesis and use of photosynthate by the invading fungal hyphae.
Currently, outbreaks of blast disease are controlled by applying expensive and toxic fungicidal chemicals such as probenazole, tricyclazole, pyroquilon and phthalide, or by burning infected crops. These methods are only partially successful since the fungus can develop resistance to the chemical agents and weed grasses may serve as a disease reservoir after burning. Clearly, improvements are needed in methods to control blast disease in rice and other cereal crops.
Transmembrane proteins belonging to the ubiquitous ATP-binding cassette (ABC) superfamily have been identified in several genera of prokaryotes and eukaryotes, including fungi. See Higgins, “ABC transporters: from microorganisms to man” Annu. Rev. Cell Biol. 8:67-113, 1992. The P-glycoproteins are a subfamily of plasma membrane-localized ABC transporters that modulate a multi-drug resistance (MDR)-related efflux of a broad range of compounds such as sugars, inorganic ions, heavy metal ions, peptides, lipids, metabolic poisons and drugs to effectively reduce the cellular accumulation of toxic compounds by efflux from the inner leaflet of the plasma membrane to the cell exterior. See Kolaczkowski et al., “In vivo characterization of the drug resistance profile of the major ABC transporters and other components of the yeast pleiotropic drug resistance network” Microb. Drug Resist. 4:143-158, 1998; Driessen et al., “Diversity of transport mechanisms: common structural principles” Trends Biochem. Sci., 25:397-401, 2000. ABC transporters play a major role in multidrug resistance (MDR), a mechanism that operates in mammalian tumor cells. The MDR subfamily of efflux pumps includes the multidrug resistance P-glycoproteins. Several MDR-type P-glycoproteins have been shown to catalyze the ATP-dependent efflux of anti-tumor agents during cancer chemotherapy. See Cole et al., “Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line” Science, 258:1650-1654, 1992; Gottesman and Pastan, “Biochemistry of multidrug resistance mediated by the multidrug transporter” Annu. Rev. Biochem., 62:385-427, 1993.
Identification and subsequent sequence comparisons of more than a hundred genes encoding ABC transporters show that the ABC proteins have one or two well-conserved nucleotide-binding folds of approximately 200 amino acid residues and the Walker A and B motifs as well as the SGG(Q) signature. Michaelis and Berkower, “Sequence comparison of yeast ATP-binding cassette proteins” Cold Spring Harbor Symp., Quant. Biol. LX:291-307, 1995. Several members of this large superfamily transport cytotoxic agents across biological membranes, reducing the intracellular level of toxins and metabolites. Driessen et al., “Diversity of transport mechanisms: common structural principles” Trends Biochem. Sci. 25:397-401, 2000.
In filamentous fungi, ABC transporter activity likely is involved in energy-dependent efflux of fungicides or phytoalexins in Aspergillus nidulans (Andrade et al., “The ABC transporter AtrB from Aspergillus nidulans mediates resistance to all major classes of fungicides and some natural toxic compounds” Microbiology, 146:1987-1997, 2000; Andrade et al., “The role of ABC transporters from Aspergillus nidulans in protection against cytotoxic agents and in antibiotic production” Mol. Gen. Genet., 263:966-977, 2000), A. fumigatus (Tobin et al., “Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus” Gene, 200:11-23, 1997), Botrytis cinerea (Vermeulen et al., “The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil” Pest Manag. Sci. 57:393-402, 2001), M. grisea (Urban et al., “An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease” EMBO J. 18:512-521, 1999), Gibberella pulicaris (Fleissner et al., “An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers” Mol. Plant-Microbe Interact. 15: 102-108, 2002) and Mycosphaerella graminicola (Stergiopoulos et al., “The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves” Mol. Plant Microbe Interact. 16:689-698, 2003; Zwiers et al., “ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds” Mol. Genet. Genomics 269:499-507, 2003). However, none of these previously known fungal transporter molecules belong to the P-glycoprotein or MDR subfamily of the ABC transporters.